This invention relates to solid phase devices useful for detecting biologically active analytes.
For many purposes, including the production of solid phase devices, it is essential to anchor ligands such as biologically active molecules to a solid support. The anchoring of such molecules to a solid support is necessary for the production of solid phase materials. These binding members are often incorporated into solid phase devices in the form of test strips. Such test strips are currently in wide use. They are frequently used by clinicians for initial screening of patients for a variety of conditions or diseases. Examples include test strips for the detection of glucose and ketone bodies in the urine in screening for diabetes, strips for the detection of occult blood in the stool in screening for early indications of cancer of the colon, and strips to detect the presence of the hormone human chorionic gonadotropin in urine as a pregnancy test. Such test strips are also now widely used by patients themselves at home for monitoring chronic conditions such as urinary sugar in diabetes. With increasing societal concern over drug abuse, an additional application of such assays has been screening urine samples for drugs of abuse such as heroin, cocaine, marijuana, or amphetamines in large-scale drugtesting programs. Such test strips must be inexpensive to make, simple to use even by untrained personnel, and reproducible in their results, avoiding false positives and false negatives.
The biologically active molecules anchored to the support can be, for example, enzymes, hormones, antibodies, or antigens. A number of supports are used for such test strips, including plastic and paper.
Other test devices can also require anchoring ligands to solid supports as part of their construction. These test devices can be hollow vessels such as test tubes or centrifuge tubes, slides for the performance of immunoassays such as single immunodiffusion, radial immunodiffusion, and rocket immunoelectrophoresis, or porous beads for affinity chromatography.
There are several methods that have been previously used to couple biologically active molecules to a solid support. Two of the prior methods used are: (i) direct covalent attachment of the biologically active molecule to the support; and (ii) passive adsorption, particularly to plastics. Although both of these methods have been widely used, they have important disadvantages, particularly for the preparation of test strips.
Direct covalent attachment of the biologically active molecule to the solid support can require difficult, time-consuming, and expensive steps to activate the solid support with a cross-linking reagent. A commonly used method, for example, involves pretreatment of the support with the cross-linking reagent glutaraldehyde. One version of this procedure, as described in P. Tijssen, "Practice and Theory of Enzyme Immunoassays," Amsterdam, Elsevier, 1985, p. 306, requires a four-hour incubation of the solid support with glutaraldehyde, several washing steps, a three-hour incubation of the solid support with the molecule to be coupled, several more washing steps, a further one-hour incubation with an amino acid to block the remaining activated sites on the solid support, and still more washing steps. The cross-linking reaction with glutaraldehyde can be difficult to control, and can result in inactivation of the biologically active molecule.
Other coupling agents than glutaraldehyde can be used for covalent attachment. Examples of these agents include carbodiimides, cyanogen bromide, and carbonyldiimidazole. These coupling agents have many of the same disadvantages as glutaraldehyde, except that the reactions can perhaps be better controlled with these reagents.
Another serious disadvantage in the use of the direct covalent attachment method is that it is often difficult to activate some widely-used solid supports, such as glass and some plastics.
The method of passive adsorption of proteins to plastics is easier to perform than covalent binding. However, it also has disadvantages. The process works at best poorly with solid supports other than plastic. In most cases, molecules other than proteins and some lipid and lipopolysaccharide antigens cannot be effectively adsorbed. Some very important classes of biologically active molecules, such as native DNA, small oligopeptides, and haptens, are at best poorly adsorbed to the plastic. A relatively long period of incubation is frequently needed, and the reagents usually must be refrigerated during this long period of incubation. Further, the adsorption of proteins onto plastic can be difficult to control. Much desorption of the bound protein can occur during subsequent incubation. The efficiency of the binding can vary unpredictably with different proteins and with different brands of plastic. Moreover, for uncertain reasons, there can be considerable variability in the binding of protein from one well on a single plate to another well. Nonspecific interactions between proteins added later during the assay process and plastic can also occur. These nonspecific interactions can diminish the reproducibility of assays carried out in such plates, and may have to be suppressed in some cases by the addition of extraneous protein, such as non-immune goat serum. These problems make the passive adsorption process unsuitable for preparation of test strips.
Other methods for attaching biologically active molecules to a solid support have been sought. Several prior workers have employed, for various purposes, covalently attaching a biologically active molecule of interest to a monomer or sol, the monomer or sol subsequently being incorporated in some manner into a polymer or gel.
Mill et al., in U.S. Pat. No. 4,003,792, issued Jan. 28, 1977, disclose chemical conjugates of acid polysaccharides and biologically active complex organic molecules, particularly conjugates capable of forming soluble sodium salts and insoluble calcium salts. Formation of the conjugate is generally effected through basic amino or phenolic hydroxyl groups of the biologically active molecule to the acid groups of the polysaccharide.
Klein et al., in U.S. Pat. No. 4,334,027, issued June 8, 1982, disclose preparation of gel beads containing an immobilized enzymatically active substance.
Marshall, in U.S. Pat. No. 4,530,900, issued July 23, 1985, and in "A New Immunoassay Separation Technique Using Reversibly Soluble Polymers," Analytical Letters 15, 1457-1465 (1982), describes the use of reversibly soluble polymers in an immunoassay. In this procedure, antibody is coupled to sodium alginate with water-soluble carbodiimide resulting in a conjugate. The conjugate can be removed from solution as desired by converting the polymer to an insoluble form by pH adjustment or addition of metal ions. Two general immunoassay procedures are also described using these conjugates.
Other methods of immobilizing biological molecules have also been described. Liotta, in U.S. Pat. No 4,446,232, issued May 1, 1984, describes an enzyme immunoassay using a two-zoned device incorporating bound antigens. This device operates by competition for the enzyme-linked antibodies between free antigen in the test sample and immobilized antigen bound to the device.
Another procedure for immobilization of biological materials was disclosed by Rosevear, in U.S. Pat. No. 4,452,892, issued June 5, 1984. Rosevear describes a process forming a hydrogel, the hydrogel comprising a biologically active material, a polymerizable material, and a viscosity enhancing agent. The polymerizable material can chemically polymerize, be cross-linked by calcium, or gel on cooling.
These prior methods can have problems in the mass production of dip sticks, such as failure to couple the iigand covalently to the polymer, failure to bind the polymer rapidly to a solid support, and difficulty in retaining the activity of the attached ligand when resolubilizing the polymer. Accordingly, none of these disclosures teaches a mass producible dip stick for biologically active analytes or a method for producing such dip sticks.
Therefore there is a need for binding members such as dip sticks that can be produced rapidly and inexpensively. These dip sticks must preserve the biological activity of the attached molecules, be usable with a wide variety of biologically active molecules and solid supports, and be sufficiently stable to be stored in the dry state.